The following points highlight the top two types of molecular maps. The types are: 1. Molecular Physical Map 2. Molecular Genetic Map.
Type # 1. Molecular Physical Map:
In molecular physical map, actual distances are measured in terms of nucleotide sequences.
(a) In Situ Hybridization (ISH):
Fluorescence in situ hybridization (FISH) and Genomic in situ hybridization (GISH):
The technique of in situ hybridization (ISH) can be used to physically map the molecular markers on well spread metaphase chromosome using corresponding probes. The probes used for hybridization with specific DNA sequences are labelled with radioactive or fluorescent compounds which can be detected through autoradiography (ISH) or fluorescence microscopy (FISH).
Physical mapping of repetitive DNA sequences and ribosomal DNA in many species has been done using this technique. If this technique is applied with total genomic probes where the plant has multi-genomic constitution, the parental chromosome can be directly identified in the hybrid, such as Triticum and Scale in the hybrid Triticale and in the hybrid of Gasteria and Aloe – thus the method is termed as GISH (Fig. 18.13).
(b) Restriction Mapping:
Determination of nucleotide sequence of a gene is done by cleavage of corresponding DNA at specific sites with the help of restriction endonucleases. These sites of cleavage can be identified and mapped to give to a restriction map. On a restriction map, is found a linear sequence of sites, each for a specific enzyme and the distances between there are measured as number of base pairs of DNA (Fig. 18.14).
The data of digestion of a DNA molecule by more than one endonucleases can be utilized to arrange the sites of breakage in a definite order. This process involves sample is also digested by a mixture of both the enzymes to confirm the results of individual successive digests. Overlapping region of different digests (A and B) can be detected which allow to prepare the restriction map.
(c) Other Methods:
Following methods are also used for this purpose:
(i) Chromosome walking:
It is a technique where different sequential clones from a genomic library are identified by successive hybridization and restriction maps are prepared separately. As a result the whole chromosomal regions are characterized and restriction map of entire chromosome can be prepared following the technique of chromosome walking.
(ii) Chromosome jumping:
Chromosome jumping approach to construct the physical map is utilized in order to bring the molecular marker close to the gene of interest. This technique will minimize the size of the DNA segments carrying the gene to be cloned in comparison to the used linked molecular marker.
(iii) Radiation hybrid mapping:
The radiation hybrids are somatic cell hybrids in which irradiated fragments of human DNA are randomly integrated into the rodent chromosomes. After screening a panel of hybrid clones with a human marker allows the marker to be mapped.
(iv) QTL mapping:
A QTL (Quantitative Trait Loci) may be defined as a region of the genome that is associated with an important quantitative agronomic trait. The QTL mapping is an advanced method using RFLP maps. It exploits the complete linkage maps by interval mapping of ‘QTL’ and identifies the crosses for QTL mapping.
The effect of each genome segment located between a pair of marker loci (but not QTL associated with a single RFLP) is assessed by interval mapping.
Type # 2.Molecular Genetic Map:
These maps are prepared utilizing the following molecular markers:
(a) Restriction Fragment Length Polymorphism (RFLP):
When the genomic DNA from several individuals (genetically related) are digested with same restriction enzymes and separated on gel through electrophoresis, blotted and hybridized with a labelled DNA clone of known sequence, polymorphism in hybridization pattern reveals the relatedness among different individuals, and such variation is termed as ‘Restriction Fragment Length Polymorphism’ (RFLP) (Fig. 18.15).
RFLP is used to infer phylogenetic relationship in both plants and animals. This technique is also used to prepare chromosome maps in human, mice, fruitfully, or in plants like maize, tomato, lettuce and rice. The inheritance or linkage relationship also can be studied through this technique analysing the parent, F1 and F2 population.
(b) Random Amplified Polymorphic DNA (RAPD):
If artificially synthesized primers with arbitrary sequences are used for PGR (Polymerase Chain Reaction) amplification, DNA segments to be amplified will be selected at random which will thus provide a truly random sample of DNA markers, which are described as ‘Random Amplified Polymorphic DNA’ (RAPD).
The steps involved are:
(i) Amplification of extracted DNA by PGR using random primers.
(ii) Separation of amplified DNA on agarose gel.
(iii) Visualization of markers on the gel and its photography.
(c) Amplified Fragment Length Polymorphism (AFLP):
This is a modified technique of RAPD, being currently used.
(d) Variable Number Tandem Repeats (VNTR):
The DNA differences between two individuals can be detected by variable number of tandem repeats which are short repeat sequences arranged in tandem order. VNTRs are divided into two groups — Minisatellites (10-25 bps) and Microsatellites (1-5 bps). A DNA poly-core probe of such micro- or mini- satellites can detect simultaneously a large number of variable loci containing tandem DNA repeat sequences.
(e) Sequence-tagged Sites (STS):
Sequence tagged sites are DNA length of 100-500 bp that are unique in genome. They are created by polymerase chain reaction amplification of primers obtained by sequencing segments of the genome.
(f) Single Nucleotide Polymorphism (SNP):
The map can also be marked off in differences among individuals that amount to changes in single base pairs. These differences are called SNPs.